I am sure my template does not have any problem, because I successfully got the correct fragments by using the primer F1 and another reverse primer R2 (about 1.2kb), as well as by using another forward primer F2 and the primer R1 (about 300bp). Also, the reason why I used 2min30s to initiate the denaturation is that I think my cDNA sample is a complex mixture. You may wondering why I use 72°C as the annealing temperature. Because I used the NEB Tm calculator online, and it gave me this answer. This annealing temperature also worked well with primer pair F1&R2.

The only different between the F1&R1 product and F1&R2 product is that the former is more than twice the size of latter. Why I already increased the extension time but was not able to get anything but some small size primer dimers. I check the primer dimer score of F1&R1 and it was not worse than F1&R2 and F2&R1. What happened to my Phusion PCR???

I appreciate your help!

-jerrywu1987-

I suspect that your annealing temperature is dramatically too high. You probably are calculating it based on the total primer length, whereas the important length is the portion which matches your template. I'd suggest an annealing temperature of 55 as a starting point.

-phage434-

phage434 on Wed Mar 5 01:10:51 2014 said:

I suspect that your annealing temperature is dramatically too high. You probably are calculating it based on the total primer length, whereas the important length is the portion which matches your template. I'd suggest an annealing temperature of 55 as a starting point.

i agree. Only the part of the primer that actually anneals to the template is used to calculate the tm.

-perneseblue-

phage434 on Wed Mar 5 01:10:51 2014 said:

I suspect that your annealing temperature is dramatically too high. You probably are calculating it based on the total primer length, whereas the important length is the portion which matches your template. I'd suggest an annealing temperature of 55 as a starting point.

Thanks for your answer. Both primers are 20~22 bp long, and all match my template. Moreover, I found the annealing temperature calculated by NEB Tm Calculator is also associated with the polymerase. If I use Taq, the annealing temperature will be 65C. But anyway, I will try temp gradient. starting from 55C.

-jerrywu1987-

Such a high annealing temperature for a primer of 22 bases probably means your GC content is very high. Lots of problems occur with very high GC. You could raise the DMSO concentration to 6%, or (better in my experience) switch from DMSO to a solution of 1 M Betaine. But I'm guessing that the gradient will fix things. This is another example of the near uselessness of Tm calculators, in my opinion. People actually believe the results, and they are rarely correct. Is there a decent GC percentage near the 3' end of the primer? High GC elsewhere and low GC at the 3' end will also often fail completely.

-phage434-

a normal phusion pcr reaction is this:

Initial Denaturation 98°C 30 seconds

25-35 Cycles

98°C - 5-10 seconds

45-72°C - 10-30 seconds

72°C - 15-30 seconds per kb

Final Extension 72°C 5-10 minutes

Hold 4-10°C

generally, the 45-72°C step you'd begin with 55°C

what program did you use to design the primers? something went very wrong somewhere.